PROJECT SUMMARY Alzheimer's Disease (AD) is characterized by the progressive accumulation of abnormally cleaved A? amyloid peptides and hyperphosphorylated tau proteins, which lead to amyloid plaques and neurofibrillary tangles, respectively. While it remains unclear what triggers these proteinopathies, several lines of evidence indicate that defects in intracellular trafficking may regulate AD pathogenesis. Importantly, emerging evidence suggests that AD-related proteins, including tau, amyloid precursor protein (APP) and A? amyloid peptides are secreted via exosomes. Despite these findings, it remains unclear what regulates the formation and packaging of exosomes, whether exosome biogenesis is functionally connected to intracellular trafficking of disease-related proteins, whether neurons and glia develop different mechanism to process these proteins, and if so, how abnormal proteostasis in neurons and glia cooperatively promotes neurodegeneration. We have discovered a new pathway in which the autophagy machinery specifies packaging and secretion of proteins within exosomes. Traditionally studied as an autodigestive pathway that promotes cell survival during stress, autophagy also promotes the unconventional secretion of proteins lacking N-terminal signal sequences. Using a proximity-based biotinylation (BioID) proteomics strategy, we have uncovered ~90 novel putative targets of autophagy-dependent secretion, including numerous proteins released within exosomes. These proteins biochemically interact with MAP1LC3B, a mammalian ATG8 isoform and autophagy regulator crucial for cargo sequestration. Based on these results, we hypothesize that the autophagy machinery mediates the LC3-dependent recruitment and packaging of specific intracellular cargo for their secretion via exosomes. Furthermore, we hypothesize that autophagy controls a delicate balance of secretion and intracellular trafficking of disease-relevant proteins in neurons and glia to promote neurodegeneration in AD and FTD. To test these predictions, we will: 1) Dissect whether and how autophagy specifies exosome packaging and secretion in normal and Alzheimer neural cell populations; and 2) Delineate how lysosomal dysfunction in AD and FTD impacts LC3-dependent exosome packaging and proteostasis. These studies are uniquely poised to define new functions for the autophagy machinery in the biogenesis and secretion of exosomes and to delineate its contributions to AD pathogenesis. This multi-PI R01 application synergistically merges the unique expertise of Dr. Jayanta Debnath in the cell biology of autophagy and Dr. Eric Huang in the molecular mechanisms of neurodegenerative diseases to address the goals of RFA-AG-17-051 by uncovering new machineries directing exosome biogenesis and the secretion of exosomal cargo molecules in AD.